Circulating tumor cells (CTC) of epithelial origin are present in the blood of carcinoma patients at very low frequency (<10/ml blood). The detection of tumor cells in circulation may have significance for cancer disease management. The detection of low frequency cells requires a large blood volume for processing. In order to enumerate and characterize CTCs from large blood volumes, the enrichment of CTCs is necessary.
Several methods are available to enrich CTCs based on size, density and antigen. The well established commercial product for enrichment of rare cells is Veridex CellSearch CTC assay. The CellSearch CTC assay uses magnetic particles conjugated to anti-epithelial cell adhesion molecule (EpCAM) to capture CTCs from 7.5 ml of blood. The enriched samples are stained with DAPI, a nucleic acid dye, to identify nucleated cells, anti-cytokeratin antibodies conjugated to phycoerythrin in order to identify cells of epithelial origin, and anti-leukocyte antibodies conjugated to allophycocyanin to identify all leukocytes. The samples are analyzed on a CellTracks Analyzer II for enumeration of CTCs.
The final sample after the enrichment contains CTCs and a small number of white blood cells (1000-5000 cells). The enrichment method removes more than 99% of white blood cells (WBC). The presence of white blood cells during the enumeration of rare cells is not an issue does not adversely affect the process. WBCs may be labeled with a leukocyte marker (CD45) to differentiate WBCs from CTCs. However, if one wants to molecularly characterize the enumerated CTCs to define their genotype by amplification of the nucleic acids present in such CTCs the presence of white blood cells in adversely affects this characterization. Given that the ratio of white cells in an enumerated fraction is high compared to the number of CTCs and the nucleic acids of such white cells is amplified with the CTCs it is difficult was difficult to determine the source of the nuclei acids, namely CTCs or white blood cells. As a result, further purification of enriched samples to remove white blood cells (decrease the ratio of white blood cells to CTCs) would be a major milestone for developing a reliable molecular characterization tools. This need is met by the following invention.
There are no methods available to remove white blood cells after the enrichment of CTCs. It requires a unique method since the same principle used for the enrichment of CTCs will not be used. For example, if the enrichment method in the first step uses the immunomagnetic method, another method besides the immunomagnetic method must be used.